Cysteine peptidases and their inhibitors in breast and genital cancer

Cysteine proteinases and their inhibitors probably play the main role in carcinogenesis and metastasis. The metastasis process need external proteolytic activities that pass several barriers which are membranous structures of the connective tissue which includes, the basement membrane of blood vessels. Activities of the proteinases are regulated by endogenous inhibitors and activators. The imbalance between cysteine proteinases and cystatins seems to be associated with an increase in metastatic potential in some tumors. It has also been reported that proteinase inhibitors, specific antibodies for these enzymes and inhibition of the urokinase receptor may prevent cancer cell invasion. Some proteinase inhibitor could serve as agents for cancer treatment.     

Modulation of the ERK pathway of signal transduction by cysteine proteinase inhibitors

Cell proliferation requires the coordinate synthesis and degradation of many proteins. In addition to the well-characterized involvement of the proteasome in the degradation of several cell cycle-regulated proteins, it has been established that cysteine proteinases are also involved in the control of cell proliferation, but their role is currently not understood. By using both synthetic cysteine proteinase inhibitors and overexpression of T-kininogen (T-KG), a physiologically relevant cysteine proteinase inhibitor, we show that inhibition of cysteine proteinases results in a severe inhibition of the ERK pathway of signal transduction. Mechanistically, this effect appears to be the result of stabilization of the ERK phosphatase MKP-1, which leads to an enhanced dephosphorylation (and hence inactivation) of ERK molecules. These results are specific to cysteine proteinase inhibitors and are not observed when either serine proteinases or the proteasome are inhibited. We hypothesize that inhibition of cysteine proteinases in vivo leads to a dysregulation of the ERK pathway, which results in an inability of the cell to transmit to the nucleus the signals generated by the presence of growth factors, thus resulting in loss of cell proliferation.     

Stimulation of high-affinity GTPase by trypsin and trypsin-like proteinases in membranes of human platelets.

The influence of various proteinases on GTP hydrolysis was studied in membranes of human platelets. Of the proteinases examined, trypsin, acrosin and a recently described trypsin-like proteinase from bovine sperm, but not chymotrypsin, increased GTP hydrolysis. Similar to what was described previously for hormone-like agents, the stimulation of GTP hydrolysis by the proteinases was only observed at low GTP concentrations, with apparent Km values of 0.2-0.3 microM-GTP. Stimulation of the high-affinity GTPase by the proteinases occurred without apparent lag phase and was constant over a long period of incubation. The proteinase inhibitors leupeptin and soya-bean trypsin inhibitor blocked the stimulation of GTP hydrolysis, but did not reverse the effect of the proteinases. Treatment of platelet membranes with N-ethylmaleimide, which eliminates Gi-protein (inhibitory guanine-nucleotide-binding protein)-related GTPase stimulation by adrenaline, decreased stimulation of GTP hydrolysis by the proteinases only partially. Activation of GTP hydrolysis by the proteinases was partially additive with that caused by adrenaline, whereas thrombin stimulation was not increased further. The data indicate that, similarly to the proteinase thrombin, trypsin and trypsin-like proteinases can activate GTP-hydrolysing protein(s) that exhibit high affinity for GTP in platelet membranes. It is suggested that the proteinases interact in platelet membranes with a receptor site similar to that used by thrombin and that the observed GTPase stimulation is a reflection of a proteinase-receptor interaction with a guanine-nucleotide-binding regulatory protein.     

Aminopeptidases as potential targets for the control of the Australian sheep blowfly, Lucilia cuprina.

A series of experiments were carried out to investigate the role of proteinase enzymes in the growth of larvae of the sheep blowfly, Lucilia cuprina. First, instar larvae were incubated on an artificial growth media in the presence of various concentrations of inhibitors of all the major proteinase classes. Inhibitors of serine proteinases and aminopeptidases were found to cause significant growth inhibition and in some cases death of the larvae within 24 h, suggesting that these enzymes were the major classes involved in protein digestion in the gut of the insect. A second group of experiments analysed the effects of two inhibitors from the same or different proteinase classes in the growth media. Synergistic inhibition of larval growth was observed with the incorporation of inhibitors of serine proteinases and aminopeptidases. The results suggest that these classes of proteinases are both central to protein digestion in this insect, probably in the gut, and that the inhibition of both types of activity leads to an almost complete blockade of digestion. Testing in vivo gave similar results with infections on sheep skin inhibited by either serine proteinase or aminopeptidase enzyme inhibitors and the combination of both stopped the infection process. The role of aminopeptidases in larval metabolism and as potential targets for blowfly control agents is examined.     

Modulation of the ERK pathway of signal transduction by cysteine proteinase inhibitors

Cell proliferation requires the coordinate synthesis and degradation of many proteins. In addition to the well‐characterized involvement of the proteasome in the degradation of several cell cycle‐regulated proteins, it has been established that cysteine proteinases are also involved in the control of cell proliferation, but their role is currently not understood. By using both synthetic cysteine proteinase inhibitors and overexpression of T‐kininogen (T‐KG), a physiologically relevant cysteine proteinase inhibitor, we show that inhibition of cysteine proteinases results in a severe inhibition of the ERK pathway of signal transduction. Mechanistically, this effect appears to be the result of stabilization of the ERK phosphatase MKP‐1, which leads to an enhanced dephosphorylation (and hence inactivation) of ERK molecules. These results are specific to cysteine proteinase inhibitors and are not observed when either serine proteinases or the proteasome are inhibited. We hypothesize that inhibition of cysteine proteinases in vivo leads to a dysregulation of the ERK pathway, which results in an inability of the cell to transmit to the nucleus the signals generated by the presence of growth factors, thus resulting in loss of cell proliferation. J. Cell. Biochem. 80:11–23, 2000. © 2000 Wiley‐Liss, Inc.     

Structural basis of the endoproteinase-protein inhibitor interaction

Proteolytic enzymes are potentially hazardous to their protein environment, so that their activity must be carefully controlled. Living organisms use protein inhibitors as a major tool to regulate the proteolytic activity of proteinases. Most of the inhibitors for which 3D structures are available are directed towards serine proteinases, interacting with the active sites in a 'canonical' i.e. substrate-like manner via an exposed reactive site loop of conserved conformation. More recently, some non-canonically binding serine proteinase inhibitors directed against coagulation factors, in particular thrombin, a few cysteine proteinase inhibitors inhibitory towards papain-like proteinases, and three zinc endopeptidase inhibitors directed against metzincins and thermolysin have been characterised in the free and complexed state, displaying novel mechanisms of inhibition with their target proteinases. These different interaction modes are presented and briefly discussed with respect to the different strategies applied by nature.     

Evidence for participation of sperm proteinases in fertilization of the solitary ascidian,Halocynthia roretzi: Effects of protease inhibitors

Effects of 15 proteinase inhibitors and an inhibitor against aminopeptidases on fertilization of the solitary ascidian, Halocynthia roretzi were studied in search of lysins. Fertilization of intact eggs was blocked by three trypsin inhibitors, leupeptin, antipain, and soybean trypsin inhibitor, and by two chymotrypsin inhibitors, chymostatin and potato proteinase inhibitor I. On the other hand, the fertilization of naked eggs was not blocked at all by leupeptin and was only partially blocked by chymostatin at the concentrations sufficient for blocking that of intact eggs. This indicates that spermatozoa utilize trypsin-like and chymotrypsin-like proteinases probably as lysins for penetrating through the chorion. The chymotrypsin-like activity appears to be also required for some step besides sperm penetration through the egg investments.     

Regulation of pro-apoptotic leucocyte granule serine proteinases by intracellular serpins

Caspase activation and apoptosis can be initiated by the introduction of serine proteinases into the cytoplasm of a cell. Cytotoxic lymphocytes have evolved at least one serine proteinase with specific pro-apoptotic activity (granzyme B), as well as the mechanisms to deliver it into a target cell, and recent evidence suggests that other leucocyte granule proteinases may also have the capacity to kill if released into the interior of cells. For example, the monocyte/granulocyte proteinase cathepsin G can activate caspases in vitro, and will induce apoptosis if its entry into cells is mediated by a bacterial pore-forming protein. The potent pro-apoptotic activity of granzyme B and cathepsin G suggests that cells producing these (or other) proteinases would be at risk from self-induced death if the systems involved in packaging, degranulation or targeting fail and allow proteinases to enter the host cell cytoplasm. The purpose of the present review is to describe recent work on a group of intracellular serine proteinase inhibitors (serpins) which may function in leucocytes to prevent autolysis induced by the granule serine proteinases.     

Effect of cysteine proteinase inhibitors on murine B16 melanoma cell invasion in vitro

Various types of proteinases are implicated in the malignant progression of human and animal tumors. Proteinase inhibitors may therefore be useful as therapeutic agents in anti-invasive and anti-metastatic treatment. The aims of this study were (1) to estimate the relative importance of proteinases in B16 cell invasion in vitro using synthetic, class-specific proteinase inhibitors and (2) to assess the inhibitory effect of some naturally occurring cysteine proteinase inhibitors. Serine proteinase inhibitor reduced invasiveness by up to 24%, whereas inhibition of aspartic proteinases reduced invasion by 11%. Synthetic inhibitors of cysteine proteinases markedly impaired invasion: cathepsin B inhibitors, particularly Ca-074Me, inhibited invasion from 20-40%, whereas cathepsin L inhibitor Clik 148 reduced invasion by 11%. The potato cysteine proteinase inhibitor PCPI 8.7 inhibited invasion by 21%, whereas another potato inhibitor, PCPI 6.6, and the mushroom cysteine proteinase inhibitor clitocypin had no effects. As the inhibitors that inhibited cathepsin B were in general more efficient at impairing the invasiveness, we conclude that of the two cysteine proteinases, cathepsin B plays a more important role than cathepsin L in murine melanoma cell invasion.     

Use of inhibitors to identify essential cysteine proteinases of Trichomonas vaginalis

Designing cysteine proteinase inhibitors as antitrichomonal drugs requires knowledge of which cysteine proteinases are essential to the parasite. In an attempt to obtain such information, the effects of a number of cysteine proteinase inhibitors on trichomonad growth in vitro and proteinase activity were investigated. The broad specificity inhibitor trans-epoxysuccinyl-l-leucylamido-(4-guanidino)butane (known as E-64) had little effect on growth of Trichomonas vaginalis (27% inhibition at 280 μM, none at 28 μM) even though the addition of 2.8 μM E-64 to growth medium resulted in inhibition of all but two (apparent molecular masses: 35 k and 49 k) of the parasite's proteinases detected by gelatin SDS-PAGE. This shows that many of the parasite's cysteine proteinases are not essential for growth in axenic culture. In contrast, a peptidyl acyloxymethyl ketone, N-benzoyloxycarbonyl-Phe-Ala-CH₂OCO-(2,6,-(CF₃)₂)Ph, at 16 μM killed T. vaginalis and severely inhibited growth of Tritrichomonas foetus. Exposure of Trichomonas vaginalis to 16 μM of this compound for 1 h resulted in both the 35 kDa and 49 kDa proteinases being inhibited, whereas some other proteinases were unaffected. Similar distinctions between the inhibitor sensitivity of the parasite's cysteine proteinases were apparent when a biotinylated peptidyl diazomethyl ketone was used to detect active proteinases. These data suggest that the growth inhibitory effects of the peptidyl acyloxymethyl ketone are through inhibition of cysteine proteinases that are not affected when the parasites are grown in the presence of E-64. At least one of these enzymes, which include the 35 kDa and 49 kDa cysteine proteinases, must be essential and so a suitable target for chemotherapeutic attack.     

Effects of proteinase inhibitors on the cutaneous lesion ofSporothrix schenckii inoculated hairless mice

Sporothrix schenckii produces two extracellular proteinases, namely proteinase I and II. Proteinase I is a serine proteinase, inhibited by chymostatin. On the other hand, proteinase II is an aspartic proteinase, inhibited by pepstatin. The addition of either pepstatin or chymostatin to the culture medium did not inhibit cell growth, however the addition of both inhibitors strongly inhibited fungal growth. Accordingly, this suggested that extracellular proteinases play an important role in cell growth and that such cell growth may be suppressed if these proteinases are inhibited. In order to substantiate this speculation in sporotrichosis, the effects of proteinase inhibitors on the cutaneous lesions of mice were studied. Ointments containing 0.1% chymostatin, 0.1% pepstatin and 0.1% chymostatin-0.1% pepstatin were applied twice daily on the inoculation sites of hairless mouse skin, and the time courses of the lesions examined. The inhibitory effect in vivo onS. schenckii was similar to that demonstrated in our previous in vitro study. Compared to the control, the time course curve of the number of nodules present after the application of either pepstatin or chymostatin was slightly suppressed. The application of both pepstatin and chymostatin, however, strongly suppressed nodule formation. This study not only confirmed the role of 2 proteinases ofS, schenckii for fungal growth in vivo, but also may lead to their use as new topical therapeutic agents.     

植物蛋白酶抑制剂在植物抗虫与抗病中的作用

综述了植物蛋白酶抑制剂抗虫与抗病作用的研究进展.蛋白酶抑制剂广泛存在于植物体内,与植物抗虫抗病密切相关.植物蛋白酶抑制剂能抑制昆虫肠道蛋白酶,使昆虫生长发育缓慢,甚至死亡.但取食蛋白酶抑制剂后,昆虫能迅速分泌对抑制剂不敏感的蛋白酶,而使蛋白酶抑制剂无效.食物蛋白的含量和质量也影响植物蛋白酶抑制剂的抗虫效果.病原菌的感染能诱导植物产生蛋白酶抑制剂,诱导产生的蛋白酶抑制剂能抑制病原菌的生长.     

Enzymatic Response of the Eucalypt Defoliator Thyrinteina arnobia (Stoll) (Lepidoptera: Geometridae) to a Bis-Benzamidine Proteinase Inhibitor

Ingestion of proteinase inhibitors leads to hyperproduction of digestive proteinases, limiting the bioavailability of essential amino acids for protein synthesis, which affects insect growth and development. However, the effects of proteinase inhibitors on digestive enzymes can lead to an adaptive response by the insect. In here, we assessed the biochemical response of midgut proteinases from the eucalypt defoliator Thyrinteina arnobia (Stoll) to different concentrations of berenil, a bis-benzamidine proteinase inhibitor, on eucalyptus. Eucalyptus leaves were immersed in berenil solutions at different concentrations and fed to larvae of T. arnobia. Mortality was assessed daily. The proteolytic activity in the midgut of T. arnobia was assessed after feeding on plants sprayed with aqueous solutions of berenil, fed to fifth instars of T. arnobia for 48?h before midgut removal for enzymatic assays. Larvae of T. arnobia were able to overcome the effects of the lowest berenil concentrations by increasing their trypsin-like activity, but not as berenil concentration increased, despite the fact that the highest berenil concentration resulted in overproduction of trypsin-like proteinases. Berenil also prevented the increase of the cysteine proteinases activity in response to trypsin inhibition.     

Effects of proteinase inhibitors on preimplantation embryos in the rat

Proteinase inhibitors of microbial origin were injected into the uterine horns of mated rats at 14:00 h on Day 5 of pregnancy (spermatozoa in vaginal smear = Day 1), and 5 or 6 h later the embryos were flushed from the horns and examined. Chymostatin and alpha-MAPI, inhibitors of chymotrypsin-like serine proteinase and thiol proteinases, as well as thiolstatin, an inhibitor of thiol proteinases, significantly inhibited embryo growth. The inhibitory activity of alpha-MAPI on embryonic growth was distinctly greater than that of thiolstatin, although the ID50 values of the two inhibitors to papain are similar. Antipain and leupeptin which are inhibitors of trypsin-like and thiol proteinases, and talopeptin, an inhibitor of metal proteinases, significantly interrupted the removal of the zona pellucida from expanding blastocysts. These results suggest that (1) a chymotrypsin-like proteinase seems to be important to the growth of the embryo, (2) a thiol proteinase may participate in embryonic growth, and (3) a trypsin-like proteinase and a metal proteinase are likely to participate in zonalysis.     

Cysteine proteinase forms in sprouting potato tuber

Transformation of plants with exogenous proteinase inhibitor genes represents an attractive strategy for the biological control of insect pests. However, such a strategy necessitates a thorough characterization of endogenous proteinases. which represent potential target enzymes for the exogenous inhibitors produced. In the present study. changes in general endoproteolytic activity were monitored during sprouting of potato ( Solanum tuberosum L. cv. Kennebec) tuber. Quantitative data obtained using standard procedures showed that an increase in cysteine proteinase (EC 3.4.22) activity occurs during sprouting. This increased activity results from the gradual appearance of new cysteine proteinase forms, as demonstrated by the use of class-specific proteinase activity gels. While only one cysteine proteinase form was present during early sprouting, at least six new active forms of the same class were shown to appear gradually after the mature tuber was sown, suggesting the involvement of a complex cysteine proteolytic system in the last stages of tuber protein breakdown. Interestingly, oryzacystatins I and II. two cysteine proleinase inhibitors potentially useful for insect control, had no effect on any tuber proteinase delected. Similar results were obtained with leaf, stem and stolon proteinases. This apparent absence of direct interference supports the potential of oryzacystatin genes for production of insect-tolerant transgenie potato plants.     

Interaction of heparin cofactor II with neutrophil elastase and cathepsin G

We investigated the interaction of the human plasma proteinase inhibitor heparin cofactor II (HC) with human neutrophil elastase and cathepsin G in order to examine 1) proteinase inhibition by HC, 2) inactivation of HC, and 3) the effect of glycosaminoglycans on inhibition and inactivation. We found that HC inhibited cathepsin G, but not elastase, with a rate constant of 6.0 x 10(6) M-1 min-1. Inhibition was stable, with a dissociation rate constant of 1.0 x 10(-3) min-1. Heparin and dermatan sulfate diminished inhibition slightly. Both neutrophil elastase and cathepsin G at catalytic concentrations destroyed the thrombin inhibition activity of HC. Inactivation was accompanied by a dramatic increase in heat stability, as occurs with other serine proteinase inhibitors. Proteolysis of HC (Mr 66,000) produced a species (Mr 58,000) that retained thrombin inhibition activity, and an inactive species of Mr 48,000. Amino acid sequence analysis led to the conclusion that both neutrophil elastase and cathepsin G cleave HC at Ile66, which does not affect HC activity, and at Val439, near the reactive site Leu444, which inactivates HC. Since cathepsin G is inhibited by HC and also inactivates HC, we conclude that cathepsin G participates in both reactions simultaneously so that small amounts of cathepsin G can inactivate a molar excess of HC. High concentrations of heparin and dermatan sulfate accelerated inactivation of HC by neutrophil proteinases, with heparin having a greater effect. Heparin and dermatan sulfate appeared to alter the pattern, and not just the rate, of proteolysis of HC. We conclude that while HC is an effective inhibitor of cathepsin G, it can be proteolyzed by neutrophil proteinases to generate first an active inhibitor and then an inactive molecule. This two-step mechanism might be important in the generation of chemotactic activity from the amino-terminal region of HC.     

Digestive proteinases in Lasioderma serricorne (Coleoptera: Anobiidae)

The cigarette beetle, Lasioderma serricorne (Fabricius), is a common pest of stored foods. A study of digestive proteinases in L. serricorne was performed to identify potential targets for proteinaceous biopesticides, such as proteinase inhibitors. Optimal casein hydrolysis by luminal proteinases of L. serricorne was in pH 8.5-9.0 buffers, although the pH of luminal contents was slightly acidic. Results from substrate and inhibitor analyses indicated that the primary digestive proteinases were serine proteinases. The most effective inhibitors of caseinolytic hydrolysis were from soybean (both Bowman Birk and Kunitz), with some inhibition by chymostatin, N-tosyl-L-phenylalanine chloromethyl ketone, and leupeptin. Casein zymogram analysis identified at least eight proteolytic activities. Activity blot analyses indicated one major proteinase activity that hydrolysed the trypsin substrate N-alpha-benzoyl-L-arginine rho-nitroanilide, and three major proteinase activities that hydrolysed the chymotrypsin substrate N-succinyl ala-ala-pro-phe rho-nitroanilide. The absence of cysteine, aspartic, and metallo proteinases in L. serricorne digestion was evidenced by the lack of activation by thiol reagents, alkaline pH optima, and the results from class-specific proteinase inhibitors. The data suggest that protein digestion in L. serricorne is primarily dependent on trypsin- and chymotrypsin-like proteinases.     

Molecular basis of Colorado potato beetle adaptation to potato plant defence at the level of digestive cysteine proteinases

Potato synthesises high levels of proteinase inhibitors in response to insect attack. This can adversely affect protein digestion in the insects, leading to reduced growth, delayed development and lowered fecundity. Colorado potato beetle overcomes this defence mechanism by changing the composition of its digestive proteinases. The induced cysteine proteinases in the adapted gut sustain a normal rate of protein hydrolysis either by inactivating the inhibitors by cleavage or by insensitivity to the inhibitors as a result of high Kis. In this study cDNA clones of cysteine proteinases in adapted guts were isolated by nested PCR on the basis of N-terminal sequences previously determined for purified enzymes (Gruden et al., 2003). The cysteine proteinase cDNAs can be classified into three groups: intestains A, B and C. The amino acid identity is more than 91% within and 35-62% between the groups. They share 43-50% identity to mammalian cathepsins S, L, K, H, J and cathepsin-like enzymes from different arthropods. Homology modelling predicts that intestains A, B and C follow the general fold of papain-like proteinases. Intestains from each group, however, differ in some specific structural characteristics in the S1 and S2 binding sites that could influence enzyme-inhibitor interaction and thus, provide different mechanisms of resistance to inhibitors for the different enzymes. Gene expression analysis revealed that the intestains A and C, but not B, are induced twofold by potato plants with high levels of proteinase inhibitors.     

Multiple acid proteinases in the cellular slime mould Dictyostelium discoideum.

Proteinase activity in the cellular slime mould Dictyostelium discoideum has been analyzed by electrophoresis on polyacrylamide gels containing denatured hemoglobin. At least eight bands due to acid proteinases have been defined using extracts of myxamoebae, four bands A-D which move faster than the fifth and major band E, a minor band E' which moves just behind E and two slow bands G and H. Fruiting body formation was accompanied by the appearance of one new proteinase band F. The proteinases were present in extracts of both axenically-grown and bacterially-grown cells. Differences between the pH dependence and stability of the individual proteinases were detected. Inhibitor studies suggested that the faster proteinases A-D may be cathepsin B-like, whilst the slower enzymes E, E' and F do not fit readily into any known group of proteinases since they were sensitive to HgCl2 but not to other inhibitors of cathepsin B and not to inhibitors of cathepsin D-like proteinases under standard conditions. None of the proteinases was apparently formed during or after preparation of extracts and the proteinases could be re-run on polyacrylamide gels to give only the band expected from the first run. The bands are believed to reflect multiple proteinase activities within the cell.     

Fluorescence methods for localizing proteinases and proteinase inhibitors in skeletal muscle

Summary Proteinases and proteinase inhibitors have become suspect in a wide variety of muscle wasting conditions that might be treatable if knowledge of the cellular locale and function of these molecules were known. Fluorescent probes have been useful in the localization of proteinases in muscle samples from human and animal specimens. These include the histochemical localization of proteinases based on the specific fluorescence of hydrolysis product derivatives, but this approach has been limited to the lysosomal proteinases because of the acidic requirements of the trapping reaction of the primary reaction product. Immunohistochemical techniques do not have the same restrictions and a number of lysosomal and nonlysosomal proteinases have been identified in muscle by this means. Unfortunately, they do not yield any information as to the activity of the enzymes. This is an important consideration since the extracellular environment contains a number of proteinase inhibitors, some of which may be internalized by the cell.